The specific detection and quantification of biological molecules has been accomplished with excellent sensitivity for example by the use of radio-labeled reporter molecules. The first radio immunoassays developed in the end of the 1950's have matured into the most important tools of in vitro diagnostics, especially in medicine, using a broad variety of different detection or reporter systems. Well-known examples of reporter molecules are enzymes, labeled latex beads, fluorescent dyes and especially chemiluminescent dyes.
Reviews describing the theory and practice of specific binding assays are available. The skilled artisan will find all necessary technical details for performing specific binding assays in textbooks like Tijssen, “Practice and theory of enzyme immunoassays” (1990) Amsterdam, Elsevier and various editions of Colowick, S. P., and Caplan, N. O., Methods in Enzymology (1980-1986), Academic Press, dealing with immunological detection methods, especially volumes 70, 73, 74, 84, 92 and 121.
Paralleled by the development of light measuring techniques and the commercial availability of highly sensitive apparatuses, luminophores have in many applications replaced isotopic labels. Some of the new luminescent labels facilitate analyte detection at extremely low levels of sensitivity. Therefore such labels also commercially are very interesting.
Luminescent labels may be subdivided into the group of fluorescent labels and the group of luminescent labels. Whereas fluorescent labels require irradiation of a sample with excitation light in order to detect and measure the fluorescent label present, the luminescent systems, e.g., chemiluminescent systems do not require an extra source of light.
A widely used class of chemiluminescent labels are the acridinium compounds. Their mechanism of chemiluminescence has been extensively studied and is nicely summarized in a review article published by Mayer, A., and Neuenhofer, S., Angewandte Chem. Intern. Ed. Engl. 33 (1994) 1044-1072, Weinheirn, VCH Verlagsgesellschaft mbH, as well as in a review article by Dodeigne, C., et al., Talanta (2000) 415-438.
Several mechanisms leading to emission of light according to the chemiluminescence principles have been proposed. Short-lived intermediates are considered part of the processes leading to decarboxylation and emission of light. The processes postulated for acridinium ester labels, resulting in emission of light or in the unwanted side reaction (dark reaction) leading to hydrolysis of the ester, are schematically shown in FIG. 1.
According to the proposed mechanism the carbonyl group (which has been part of the amide or ester bond) by attack of H2O2 becomes part of a dioxetanone moiety. Spontaneous decomposition of the dioxetanone moiety is accompanied by light emission and yields a heterocyclic ketone and CO2 in case of a carbonyl group, or in more general chemical terms a heterocumulene in case functional equivalents of the carbonyl group had been present.
It is instantly evident from FIG. 1, that the light reaction (LR) and the dark processes (DP) both are dependent on the properties of the leaving group Z.
An essential feature of the acridinium esters used in diagnostic applications is that the ester function has been substituted to carry a suitable leaving group Z. Suitable leaving groups are designed to match as good as possible two essential requirements: stability and high quantum yield.
On the one hand the leaving group of an acridinium esters must be as active as possible, i.e., leaving quite readily under measurement conditions, to allow for a sensitive detection and high quantum yield. This high activity on the other hand, however, goes to the expense of instability towards hydrolysis. Such instabilities are even more critical if such chemiluminescent labels are used for conjugation to biomolecules. The goal to achieve a high chemiluminescence yield and in addition a high stability of the labeled reagent equals to a fine balance act always ending in a compromise between light yield and stability.
To at least partially reduce the problems encountered, new and different leaving groups have been designed and proposed.
EP 617 288 gives examples of appropriate leaving groups. Most popular are N-sulfonamides, e.g., described in U.S. Pat. No. 5,669,819, thioesters as described in DE 3 645 292, hydroxamic acid esters described in WO 98/56765, imidazolides as described by Waldrop III, A. A., et al., Luminescence 15 (2000) 169-182, and pyridinium amides (WO 95/19976).
Besides the acridinium labels, other well known chemiluminescence based systems make use of labels comprising amongst others the following categories, the combination of luciferins with corresponding luciferases, cyclic arylhydrazides, acridinium derivatives, stable dioxetanes, and oxalic acid derivatives.
However, overall only a rather limited number of chemiluminescent basic compounds is known and even less have proven useful for routine diagnostic applications.